Genotypic Homogeneity in Distinctive Transforming Growth Factor-Beta Induced (TGFBI) Protein Phenotypes.

Background: To evaluate the distribution of the transforming growth factor-beta induced (TGFBI) corneal dystrophies in a multi-ethnic population in Singapore, and to present the different phenotypes with the same genotype. Methods: This study included 32 patients. Slit lamp biomicroscopy was performed for each patient to determine the disease phenotype. Genomic DNA was extracted from the blood samples and the 17 exons of the TGFBI gene were amplified by PCR and sequenced bi-directionally for genotype analysis. Results: Regarding phenotypes, the study patients comprised 11 (34.4%; 8 with R555W and 3 with R124H mutation) patients with granular corneal dystrophy type 1 (GCD1), 6 (18.8%; 5 with R124H and 1 with R124C mutation) patients with GCD2, 13 (40.6%; 7 with R124C, 2 with H626R, 2 with L550P, 1 with A620D and 1 with H572R) patients with lattice corneal dystrophy (LCD) and 2 (6.3%; 1 with R124L and 1 with R124C) patients with Reis-Bückler corneal dystrophy. Regarding genotype, R124H mutation was associated with GCD2 (5 cases; 62.5%) and GCD1 (3 cases; 37.5%). R124C mutation was associated with LCD (7 cases; 87.5%) and GCD2 (1 case; 12.5%). All the 8 cases (100%) of R555W mutation were associated with GCD1. Conclusions: Although the association between genotype and phenotype was good in most cases (65.7%; 21 of 32 patients), genotype/phenotype discrepancy was observed in a significant number.

A unique PRDM13-associated variant in a Georgian Jewish family with probable North Carolina macular dystrophy and the possible contribution of a unique CFH variant.

Purpose: North Carolina macular dystrophy (NCMD) is an autosomal dominant maculopathy that is considered a non-progressive developmental disorder with variable expressivity. Our study aimed to clinically and genetically characterize macular dystrophy in a family (MOL1154) consisting of six affected subjects with a highly variable maculopathy phenotype in which no correlation between age and severity exists. Methods: Clinical characterization included visual acuity testing and electroretinography. Genetic analysis included Sanger sequencing and whole exome sequencing (WES). Results: WES analysis performed on DNA samples from two individuals revealed a heterozygous deletion of six nucleotides [c.2247_2252del; p.(Leu750_Lys751del)] in the CFH gene. Co-segregation analysis revealed that five of the six NCMD affected subjects carried this deletion, while one individual who had a relatively mild phenotype compatible with dry age-related macular degeneration (AMD) did not carry it. We subsequently analyzed the upstream region of PRDM13 that has previously been reported to be associated with NCMD and identified a unique heterozygous transversion (chr6:100040974A>C) located within the previously described suspected control region in all six affected individuals. This transversion is likely to cause NCMD. Conclusions: NCMD has a wide spectrum of clinical phenotypes that can overlap with AMD, making it challenging to correctly diagnose affected individuals and family members. The DNA sequence variant we found in the CFH gene of some of the affected family members may suggest some role as a modifier gene. However, this variant still does not explain the huge phenotypic variability of NCMD and needs to be studied in other and larger populations.

Genetic analysis of CHST6 and TGFBI in Turkish patients with corneal dystrophies: Five novel variations in CHST6.

PURPOSE: To identify pathogenic variations in carbohydrate sulfotransferase 6 (CHST6) and transforming growth factor, beta-induced (TGFBI) genes in Turkish patients with corneal dystrophy (CD). METHODS: In this study, patients with macular corneal dystrophy (MCD; n = 18), granular corneal dystrophy type 1 (GCD1; n = 12), and lattice corneal dystrophy type 1 (LCD1; n = 4), as well as 50 healthy controls, were subjected to clinical and genetic examinations. The level of antigenic keratan sulfate (AgKS) in the serum samples of patients with MCD was determined with enzyme-linked immunosorbent assay (ELISA) to immunophenotypically subtype the patients as MCD type I and MCD type II. DNA was isolated from venous blood samples from the patients and controls. Variations were analyzed with DNA sequencing in the coding region of CHST6 in patients with MCD and exons 4 and 12 in TGFBI in patients with LCD1 and GCD1. Clinical characteristics and the detected variations were evaluated to determine any existing genotype-phenotype correlations. RESULTS: The previously reported R555W mutation in TGFBI was detected in 12 patients with GCD1, and the R124C mutation in TGFBI was detected in four patients with LCD1. Serum AgKS levels indicated that 12 patients with MCD were in subgroup I, and five patients with MCD were in subgroup II. No genetic variation was detected in the coding region of CHST6 for three patients with MCD type II. In other patients with MCD, three previously reported missense variations (c. 1A>T, c.738C>G, and c.631 C>T), three novel missense variations (c.164 T>C, c.526 G>A, c. 610 C>T), and two novel frameshift variations (c.894_895 insG and c. 462_463 delGC) were detected. These variations did not exist in the control chromosomes, 1000 Genomes, and dbSNP. CONCLUSIONS: This is the first molecular analysis of TGFBI and CHST6 in Turkish patients with different types of CD. We detected previously reported, well-known hot spot mutations in TGFBI in the patients with GCD1 and LCD1. Eight likely pathogenic variations in CHST6, five of them novel, were reported in patients with MCD, which enlarges the mutational spectrum of MCD.

Generation and characterization of a murine model of Bietti crystalline dystrophy.

PURPOSE: Bietti crystalline dystrophy (BCD) is a rare, autosomal recessive, progressive, degenerative eye disease caused by mutations in the CYP4V2 gene, for which no treatments are currently available. Cyp4v3 is the murine ortholog to CYP4V2, and to better understand the molecular pathogenesis of this disease we have established a Cyp4v3-null mouse line. METHODS: Cyp4v3(-/-) mice were generated by homologous recombination in embryonic stem cells. Ocular morphologic characteristics were evaluated via fundus imaging, plasma lipid profiling, and histologic analysis via Oil Red O reactivity, hematoxylin and eosin staining, and transmission electron microscopy. RESULTS: The Cyp4v3(-/-) mouse recapitulates the characteristic features of corneoretinal crystal accumulation and systemic dyslipidemia seen in BCD. The Cyp4v3(-/-) mice behave normally and are viable and fertile when maintained under specific pathogen-free (SPF) housing conditions. CONCLUSIONS: Cyp4v3(-/-) mice represent a promising preclinical model that may be used to better understand the disease etiology and to evaluate pharmacotherapies for this devastating condition.

Positive association between plasma amylin and cognition in a homebound elderly population.

Our recent study reported that amylin, a pancreatic peptide that readily crosses the blood-brain barrier, improves learning and memory in Alzheimer's disease mouse models. However, the relationship between peripheral amylin and cognition in humans is unknown. In this follow-up study, using a cross-sectional, homebound elderly population, improvement in cognitive function with increasing quartiles of plasma amylin was suggested by positive association with verbal memory (p = 0.0002) and visuoconstruction tasks (p = 0.004), and inverse association with timed measures of attention (p < 0.0001) and executive function (p = 0.04). After adjusting for demographic information, apolipoprotein E4 allele, diabetes, stroke, kidney function, and lipid profile, log10 of plasma amylin remained associated with these cognitive domains. In contrast, plasma amyloid-ß peptide was not associated with these specific cognitive domains. Our study suggests that peripheral amylin may be protective for cognitive decline, especially in the domains affected by Alzheimer's disease.

Structural collagen alterations in macular corneal dystrophy occur mainly in the posterior stroma.

PURPOSE: Collagen fibrils in the corneal stroma in macular corneal dystrophy, on average, are more closely spaced than in the normal cornea. This study was conducted to investigate if this occurs uniformly across the stroma or is more prevalent at certain stromal depths. METHODS: Microbeam synchrotron X-ray fiber diffraction patterns were obtained in 25 microm steps across the whole thickness of a thin strip of a macular corneal dystrophy cornea obtained at keratoplasty. Data were analyzed for mean collagen interfibrillar spacing at all positions. Serum was analyzed immunochemically to determine immunophenotype, and transmission electron microscopy was carried out to visualize stromal ultrastructure. RESULTS: Keratan sulphate was not detectable in blood serum, classifying the disease as macular corneal dystrophy type I. Collagen interfibrillar spacing dropped linearly with stromal depth from the anterior to posterior cornea, measuring 5-10% less in the posterior 100 microm of the MCD stroma compared to the anterior 100 microm (p < 0.001). Isolated pockets of collagen fibrils with unusually large diameters were identified in the deep stroma. CONCLUSIONS: Collagen fibril spacing is reduced and large-diameter collagen fibrils are seen in macular corneal dystrophy type I, with the deep stroma affected more. We speculate that the ultrastructural abnormalities are more prevalent in the posterior stroma because the structural influence of sulphated keratan sulphate glycosaminoglycans/proteoglycans is high in this region of the cornea.

Molecular genetic study of Egyptian patients with macular corneal dystrophy.

AIM: To identify the underlying genetic defect in Egyptian patients with macular corneal dystrophy (MCD). METHODS: A clinical and molecular genetic study was performed on 11 patients from six families with MCD. Clinical diagnosis was confirmed by slit-lamp biomicroscopy and histopathological examination of corneal buttons following keratoplasty. The coding region of the carbohydrate sulfotransferase (CHST6) gene was amplified by polymerase chain reaction (PCR) in all affected subjects. This was followed by direct sequencing and restriction digest analyses. Enzyme-linked immunosorbent assay of antigenic keratan sulfate (KS) in patients' serum was also performed. RESULTS: Six homozygous mutations, of which three are novel, were identified within the coding region of CHST6 in six unrelated MCD families. The barely detectable level of antigenic KS in the serum of the affected individuals indicated that they all have MCD type I, including the subtype IA. CONCLUSIONS: This is the first report of a molecular genetic analysis of MCD in the Egyptian population. These data indicate the extensive allelic heterogeneity within CHST6 and further support its essential role in maintaining corneal transparency.

Morphological evaluation of Schnyder's crystalline corneal dystrophy by laser scanning confocal microscopy and Fourier-domain optical coherence tomography.

A 29-year-old Chinese woman with Schnyder's crystalline corneal dystrophy was referred to our clinic. Ocular examination revealed abnormal deposits of cholesterol and lipid within the corneal stroma, appearing as crystalline needle-shaped deposits, in both eyes. In vivo laser scanning confocal microscopy revealed pathological alterations of the normal corneal anatomy in the patient, such as highly reflective crystalline structures in the anterior-and mid-stroma. The images acquired from Fourier-domain optical coherence tomography indicated that the main presence of crystalline deposits was localized within the anterior stroma, extending from the basal epithelium layer to a depth of 80 to 150 microm. To the best of the author's knowledge, it is consistent with the results of the previous histological study.

Schnyder corneal dystrophy.

PURPOSE OF REVIEW: The present review of Schnyder corneal dystrophy (SCD) corrects the misconceptions in the published literature about this disease. Understanding the clinical findings facilitates diagnosis of the dystrophy. RECENT FINDINGS: Retrospective case series of 115 affected individuals from 34 SCD families identified since 1989 reports the clinical findings in a large cohort and the long-term visual morbidity of SCD. The configuration of the progressive corneal clouding is predictable on the basis of age and, contrary to many older publications, only 54% of affected patients were found to have corneal crystals. Penetrating keratoplasty was reported in 20 of 37 (54%) patients of at least 50 years and 10 of 13 (77%) patients of at least 70 years. Best corrected visual acuity 1 year prior to penetrating keratoplasty in 15 eyes (nine patients) ranged from 20/25 to 20/400 including seven eyes with other ocular disorders. Best corrected visual acuity in the remaining eight eyes was 20/25 to 20/70. These patients often complained of glare preoperatively, which most likely resulted from light scattering from the corneal cholesterol. CONCLUSION: The literature on SCD must be changed to reflect new information about the disease. When present, corneal crystals facilitate disease diagnosis but the examiner must be aware that they are only present 54% of the time. Although scotopic vision remains good until old age, disproportionate loss of photopic vision with frequent complaints of glare necessitates penetrating keratoplasty in the majority of patients over 50 years of age.

An unusual presentation of macular corneal dystrophy associated with uniparental isodisomy and a novel Leu173Pro mutation.

PURPOSE: To report an unusual phenotype of macular corneal dystrophy (MCDC1) associated with a novel CHST6 mutation transmitted via maternal isodisomy. METHODS: Slit lamp examination of the patient and his parents was performed. DNA was collected from each individual for amplification and sequencing of the CHST6 coding region, as well as exons 4 and 12 of TGFBI. Serum antigenic keratan sulfate (AgKS) levels were measured for confirmation of the diagnosis and subtyping of MCDC1. Quantitative real-time PCR (qPCR) was performed to differentiate between homozygous and hemizygous sequence variants. Genotyping at 12 single nucleotide polymorphisms (SNPs) within and surrounding CHST6 was performed to determine the pattern of inheritance of mutations identified in CHST6. RESULTS: Examination of the proband revealed bilateral, discrete, axially distributed, gray-white deposits at the level of Bowman's layer, with diffuse fine corneal stromal haze. Screening of TGFBI exons 4 and 12 in the proband did not reveal any allelic variants. However, screening of CHST6 in the proband demonstrated a novel homozygous missense mutation involving a highly conserved amino acid (c.518T > C; Leu173Pro) and undetectable serum AgKS levels in the proband confirmed the diagnosis of type I MCDC1. Quantitative PCR confirmed that both copies of CHST6 were present in the patient, excluding the possibility that the mutation was present in the hemizygous state. The results of genotyping were consistent with maternal isodisomy, as the patient was homozygous for an allele possessed by his mother at each SNP, two of which were informative and demonstrated nonpaternal inheritance. CONCLUSION: A phenotypically unusual variant of MCDC1 was found to be associated with the novel Leu173Pro mutation in CHST6, transmitted via uniparental isodisomy, a previously unreported pattern of inheritance in the corneal dystrophies.

Central corneal mosaic opacities in Schnyder's crystalline dystrophy.

PURPOSE: To report an unusual presentation of Schnyder's corneal crystalline dystrophy (SCCD), sharing the feature of central corneal mosaic opacities. DESIGN: Observational case report. METHODS: A 51-year-old man and his family members were examined. Investigations included slit-lamp biomicroscopy, radiography of knee joint, plasma lipid level, and genotyping of the SCCD candidate region in chromosome 1p34.1-1p36. RESULTS: A symmetric, central, disciform, full-thickness opacity was seen in both corneas of the patient. The opacities appeared in a mosaic pattern, instead of collections of crystals or a diffuse haze as typically detected in SCCD. Small clumps of crystalline deposits and arcus lipoides were also observed. Systemically, hyperlipidemia and bilateral genu valgus were identified. He had 2 daughters, and both of them had bilateral corneal crystalline deposits and genu valgus. No other family members had findings suggesting SCCD. The genetic study demonstrated that all of the affected individuals shared a common haplotype within the region of previously reported SCCD locus. However, 1 unaffected sibling of the proband also had the same haplotype. CONCLUSIONS: Central corneal mosaic opacities may be another variant of SCCD.

Novel CHST6 nonsense and missense mutations responsible for macular corneal dystrophy.

PURPOSE: To identify the underlying mutations in two unrelated British families with macular corneal dystrophy (MCD) by screening the carbohydrate sulfotransferase (CHST6) gene. DESIGN: Case reports and results of DNA analysis. METHODS: Two subjects from two British families with MCD were studied. The genetic status of CHST6 was determined for all members of these MCD families. In addition, sulfated keratan sulfate (KS) assay from the probands was also undertaken. CHST6 gene was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by sequencing and restriction digestion. Enzyme-linked immunosorbent assay (ELISA) was performed to assess KS presence in serum. RESULTS: Four compound heterozygous mutations were identified, three of which are novel. The ELISA showed that the probands were of MCD type I. CONCLUSIONS: These novel mutations are expected to result in loss of CHST6 function, which would account for the MCD phenotype.

[Schnyder's crystalline corneal dystrophy. Further narrowing of the linkage interval at chromosome 1p34.1-p36?]. / Schnydersche kristalline Hornhautdystrophie. Weitere Eingrenzung des Kopplungsintervalls auf Chromosom 1p34.1-p36?

BACKGROUND: Schnyder's crystalline corneal dystrophy (SCCD) is a rare autosomal dominant disease and can occur in association with hyperlipoproteinemia. The disease has been mapped to chromosome 1p34.1-p36. CASE REPORT: We report on a 66-year-old woman and her son with Schnyder's crystalline corneal dystrophy. The mother had type IV hyperlipoproteinemia and hypercholesterolemia while her son had hypercholesterolemia with elevated LDL-cholesterol. Analysis of microsatellite markers within the candidate interval of 1p34.1-p36 showed that the affected son and his unaffected brother had inherited different alleles only for the proximal marker D1S228 from their affected mother. CONCLUSIONS: The haplotype analysis suggests that either recombination has occurred, which would allow the candidate interval to be narrowed down, or alternatively, the SCCD in the reported family is not linked to chromosome 1, which would be a first indication of genetic heterogeneity in this disease. To reduce the risk of cardiovascular disease, hyperlipidemia should always be excluded in patients with Schnyder's crystalline corneal dystrophy.

Novel mutations in the CHST6 gene associated with macular corneal dystrophy in southern India.

OBJECTIVE: To further characterize the role of the carbohydrate sulfotransferase (CHST6) gene in macular corneal dystrophy (MCD) through identification of causative mutations in a cohort of affected patients from southern India. METHODS: Genomic DNA was extracted from buccal epithelium of 75 patients (51 families) with MCD, 33 unaffected relatives, and 48 healthy volunteers. The coding region of the CHST6 gene was evaluated by means of polymerase chain reaction amplification and direct sequencing. Subtyping of MCD into types I and II was performed by measuring serum levels of antigenic keratan sulfate. RESULTS: Seventy patients were classified as having type I MCD, and 5 patients as having type II MCD. Analysis of the CHST6 coding region in patients with type I MCD identified 11 homozygous missense mutations (Leu22Arg, His42Tyr, Arg50Cys, Arg50Leu, Ser53Leu, Arg97Pro, Cys102Tyr, Arg127Cys, Arg205Gln, His249Pro, and Glu274Lys), 2 compound heterozygous missense mutations (Arg93His and Ala206Thr), 5 homozygous deletion mutations (delCG707-708, delC890, delA1237, del1748-1770, and delORF), and 2 homozygous replacement mutations (ACCTAC 1273 GGT, and GCG 1304 AT). One patient with type II MCD was heterozygous for the C890 deletion mutation, whereas 4 possessed no CHST6 coding region mutations. CONCLUSION: A variety of previously unreported mutations in the coding region of the CHST6 gene are associated with type I MCD in a cohort of patients in southern India. CLINICAL RELEVANCE: An improved understanding of the genetic basis of MCD allows for earlier, more accurate diagnosis of affected individuals, and may provide the foundation for the development of novel disease treatments.

Truncating mutations in the carbohydrate sulfotransferase 6 gene (CHST6) result in macular corneal dystrophy.

PURPOSE: Identification of mutations in the CHST6 gene in 15 patients from 11 unrelated families affected with recessive macular corneal dystrophy (MCD). METHODS: Genomic DNA was extracted from peripheral blood leukocytes of the affected patients and their healthy family members, and the mutational status of the CHST6 gene was determined for each patient by a PCR-sequencing approach. Serum concentrations of antigenic keratan sulfate for each proband were determined by ELISA. RESULTS: ELISA indicated that all affected patients, except one, were of MCD type I or IA. Fourteen distinct mutations were identified within the CHST6 coding region: 2 nonsense, 2 frameshift, and 10 missense. Of these, 12 were novel, and a nonsense mutation in the homozygous state is reported for the first time. CONCLUSIONS: These molecular results in French patients with MCD combined with those reported in previous studies indicated CHST6 mutational heterogeneity. The characterization herein of nonsense mutations is in keeping with the fact that MCD results from loss of function of the CHST6 protein product.

Measurement of activities of human serum sulfotransferases which transfer sulfate to the galactose residues of keratan sulfate and to the nonreducing end N-acetylglucosamine residues of N-acetyllactosamine trisaccharide: comparison between normal controls and patients with macular corneal dystrophy.

Human serum sulfotransferase activities were measured in normal controls and patients with macular corneal dystrophy (MCD), an inherited disorder characterized by the decreased sulfation of keratan sulfate in the corneal stroma and serum, using two kinds of acceptor: partially desulfated keratan sulfate and a trisaccharide with a GlcNAc residue at the nonreducing terminal, GlcNAcbeta1-3Galbeta1-4GlcNAc. When partially desulfated keratan sulfate was used as the acceptor, only sulfotransferase activity which transfers sulfate to position 6 of the Gal residues was detected. In contrast, when GlcNAcbeta1-3Galbeta1-4GlcNAc was used as the acceptor, sulfotransferase activity which transfers sulfate to position 6 of the nonreducing terminal GlcNAc residue could be detected. Although keratan sulfate levels in the sera of MCD patients determined by ELISA were much lower than those in normal controls, there were no detectable differences in either the sulfotransferase activity responsible for the sulfation of position 6 of Gal residues or that responsible for the sulfation of position 6 of nonreducing end GlcNAc residues between normal controls and MCD patients. These results suggest that the sulfotransferase involved in the sulfation of keratan sulfate, which is assumed to be deficient in MCD patients, may not be secreted into the serum, and that direct measurement of the sulfotransferase activity present in affected tissues such as the cornea instead of serum may be necessary to confirm the postulated deficiency in the biosynthesis of keratan sulfate in MCD.

Schnyder crystalline dystrophy sine crystals. Recommendation for a revision of nomenclature.

PURPOSE: To determine the percentage of patients with Schnyder crystal line dystrophy who had corneal crystal deposition. METHODS: Thirty-three patients with Schnyder crystalline dystrophy were identified by the author since 1987. Each patient had a complete ophthalmic evaluation, including slit-lamp examination by the author. RESULTS: Only 51% (17 of 33) of patients with Schnyder crystalline corneal dystrophy actually had clinical evidence of corneal crystalline deposits. CONCLUSIONS: Because of the confusing nomenclature, many ophthalmologists presume that the presence of corneal crystals is an integral part of the diagnosis of Schnyder crystalline dystrophy. The clinician should be aware that despite the fact that the noncrystalline form of the dystrophy has been poorly recognized, it is equally common.

Macular corneal dystrophy: reduction in both corneal thickness and collagen interfibrillar spacing.

The interfibrillar spacing of collagen fibrils was measured at twenty different positions across a macular dystrophy cornea using synchrotron X-ray diffraction. Unlike previous work of this type the cornea had not been frozen for storage. The spacings were all significantly lower than the spacings which existed at similar positions across a normal adult human cornea. This close-packing of collagen fibrils seems to be responsible for the reduced thickness of the central cornea in macular dystrophy. Neither the patient's serum or corneal tissue contained appreciable amounts of sulfated keratan sulfate, this classifies the disease as Type I macular corneal dystrophy.